This project has two primary aims. The first aim is to understand how proteins that are destined to travel through the secretory pathway are targeted to transport sites in the endoplasmic reticulum (ER) or the bacterial inner membrane (IM). For the last decade we have been investigating the role of a ribonucleoprotein called the signal recognition particle (SRP) and its membrane-bound receptor in this process. Although SRP was initially believed to exist only in eukaryotic cells, the sequencing of a large number of microbial genomes has demonstrated that the particle is found in most (if not all) organisms. Previous studies have shown that in mammalian cells SRP recognizes the "signal sequences" found on virtually all secreted and membrane proteins as they emerge during translation and then catalyzes their translocation across the ER membrane upon interaction with the SRP receptor. Several years ago we demonstrated that bacterial SRP has a somewhat more restricted function in that it only targets integral membrane proteins to the IM. Consistent with the work of other laboratories, we found that most secreted proteins, by contrast, are targeted to the IM posttranslationally by molecular chaperones. In recent studies we have continued to analyze protein targeting mechanisms and protein targeting signals in bacteria. In the past year we have focused on the targeting of a class of very large (~100-400 kD) secreted toxins called autotransporters. Autotransporters are proteins that contain a small C-terminal ?beta domain? that facilitates translocation of a large N-terminal ?passenger domain? across the outer membrane (OM) by an unknown mechanism. These proteins are produced by a wide range of pathogenic Gram negative bacteria and often contain exceptionally long signal peptides that are distinguished by a unique N-terminal extension. We recently found that the presence of the signal peptide extension promotes the targeting of a model autotransporter produced by E. coli O157:H7 (?EspP?) to the IM via a novel posttranslational mechanism. Interestingly, we also found that the unusual signal peptide was essential for late stages of protein biogenesis that occur after the protein is translocated across the IM. A second aim of the project is to elucidate the mechanism of autotransporter secretion. Using Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods we showed that the EspP beta domain behaves as a compact monomer and forms a channel that is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain attached to the N-terminus of EspP was efficiently translocated across the OM and that the native EspP passenger domain folds at least partially in the periplasm. These apparently paradoxical data strongly suggest that an external factor transports the passenger domain across the OM and that the beta domain functions primarily as a membrane anchor. Our results challenge the prevailing view of the autotransporter beta domain as an autocatalytic protein translocase and provide new insights into the biogenesis of a large class of proteins that play important roles in bacterial pathogenesis.